1. Field of the Invention
This invention relates to a truncated form of 1,3-1,4- -D-Glucanase (lichenase, EC 3.2.1.73) with enhanced enzymatic activity and thermal tolerance.
2. Description of the Related Art
1,3-1,4-β-D-Glucanase is an endo-β-D-glucanase that can specifically hydrolyze 1,4-β-D-glucosidic bonds adjacent to 1,3-β-linkages in mix-linked β-glucans, yielding mainly cellobiosyltriose and cellotriosyltetraose. The enzyme has received much attention in both basic and applied research areas because of its enzymatic functions and importance in industrial applications. Supplementation of this fibrolytic enzyme in animal feed is one of the approaches for increasing the feed conversion efficiency and growth-rate of non-ruminal animals (Bedford et al., 1992; Selinger et al., 1996). This enzyme is also attractive for its application in the brewing industry. This enzyme has been used to substitute or supplement malt enzymes for reducing the industrial processing problem(s) caused by β-glucans from cell walls of the starchy seed endosperm, which include, for example, the reduced yield of extract, lowered rates of wort separation and beer filtration, formation of hazes and gelatinous precipitates in beer (Uhilg, 1998). However, the wide use of 1,3-1,4-β-D-glucanase as an industrial enzyme in general has a major drawback, that is, the limitation imposed by the thermal stability of the enzyme during industrial processes. For instance, the elevated temperatures employed in the malting process (50–70° C.) or the feed-pelleting and/or expansion processes (65–90° C.) may cause severe inactivation of the enzyme. Therefore, creation of heat-resistant enzymes would overcome the aforementioned problem. Moreover, an enzyme with high catalytic activity would be more desirable in industrial applications in terms of cost-effectiveness.
Fibrobacter succinogenes is a microorganism that plays a major role in plant fiber digestion in the rumen. From this organism, several enzymes related to the digestion of cellulose or hemicellulose polymers of plant cell wall have been isolated and studied (Selinger et al., 1996). One of the Fibrobacter succinogenes emzymes, 1,3-1,4-β-D-glucanase or Fsβ-glucanase, is isolated and characterized by Teather et al. (1988 & 1990). This enzyme consists of a protein sequence with circular permutation in which two highly conservative catalytic domains of the enzyme are in a reverse orientation, as compared to that of 1,3-1,4-β-D-glucanases from other sources (Teather & Erfle, 1990; Schimming et al., 1992; Heinemann & Hahn, 1995). Five repeated serine-rich regions are found in the C-terminal, which are nonhomologous relative to bacilli or other bacterial 1,3-1,4-β-D-glucanases.
One objective of the present invention is to provide a new form of glucanase having both enhanced enzymatic activity and improved thermal stability. This objective is achieved by truncating a wild-type 1,3-1,4-β-D-glucanase whereby producing a shortened form of the enzyme. This truncated form of glucanase, with significant enhancement both in the enzymatic activity and in the thermal stability, and the method for producing the truncated enzyme are hereby disclosed.